This invention relates to tissue-specific gene promoters, and particularly relates to a promoter which is responsive to the root knot nematode.
A promoter is a DNA sequence which flanks a transcribed gene, and to which RNA polymerase must bind if it is to transcribe the flanking gene into messenger RNA. A promoter may consist of a number of different regulatory elements which affect a structural gene operationally associated with the promoter in different ways. For example, a regulatory gene may enhance or repress expression of an associated structural gene, subject that gene to developmental regulation, or contribute to the tissue-specific regulation of that gene. Modifications to promoters can make possible optional patterns of gene expression, using recombinant DNA procedures. See, e.g., Old and Primrose, Principles of Gene Manipulation (4th Ed., 1989).
U.S. Pat. No. 5,459,252 to Conkling and Yamamoto describes a root specific promoter designated RB7, which was identified in tobacco. U.S. Pat. No. 5,837,876 to Conkling et al. describes a root cortex specific gene promoter designated the RD2 promoter, which was also identified in tobacco.
Rather than use a promoter that is constitutively active, it is desireable to have promoters that are responsive to particular stimuli. In particular, if a promoter is responsive to a particular pathogen, then that promoter could be used to impart selective disease resistance to that pathogen through expression of a transgene that disrupts that pathogen.
U.S. Pat. No. 5,750,386 to Conkling, Opperman and Taylor describes pathogen resistant transgenic plants in which a nematode-responsive element is operatively associated with a nucleotide of interest (in this case, a gene encoding a product toxic to plant cells). One nematode responsive element was a deletion fragment of the RB7 root specific promoter described above.
U.S. Pat. No. 5,589,622 to Gurr et al. suggests nematode resistant transgenic plants in which cells of the plant contain a heterologous construct comprising a nematode responsive promoter operatively associated with a product disruptive of nematode attack. However, the DNAs disclosed by Gurr et al. as nematode responsive promoters do not appear to represent such promoters, and instead appear to represent extraneous or irrelevant DNA.
To impart useful traits to plants by the expression of foreign genes using genetic engineering techniques, a variety of pathogen-responsive promoters will be required to allow traits to be expressed selectively, in the appropriate plant tissues, and at the appropriate times. Accordingly, there is a continued need for pathogen responsive elements that operate in plant cells.
The present invention is based on the discovery that the elongation specific endo-1,4-xcex2-glucanase (cel1) promoter of Arabidopsis thaliana, described in U.S. Pat. No. 6,005,092 to O. Shosoyev and Z. Shani (Dec. 21, 1999), and Shani et al., Plant Molecular Biology 34, 837-842 (1997), is upregulated in root-knot nematode feeding cells (i.e., giant cells). Plant parasitic nematodes cause approximately 100 billion dollars annually in crop loses worldwide. The root knot nematode has a host range of over 2000 plant species, and is one of the most damaging nematodes.
Accordingly, a first aspect of the present invention is an isolated DNA molecule which directs root knot nematode responsive transcription of a downstream heterologous DNA segment in a plant cell (i.e., a promoter), and the use thereof in providing or imparting nematode resistance to plants and plant cells.
A further aspect of the present invention is construct comprising a promoter as described above and a heterologous DNA segment (i.e., a DNA segment not naturally associated with that promoter) positioned downstream from, and operatively associated with, the promoter. The heterologous DNA segment preferably encodes a product disruptive of nematode attack (i.e., a product that hinders or interferes with the ability of a nematode to feed upon a plant cell, or establish a feeding site in relationship to a plant cell, when that product is expressed in a plant cell).
Further aspects of the present invention are plant cells containing the above described constructs, methods of making transformed plants from such plant cells, the transformed plants comprising such transformed plant cells, and the use of the foregoing to impart resistance to root knot nematodes to plants.